This invention relates generally to the field of cell biology of embryonic cells and liver cells. More specifically, this invention relates to the directed differentiation of human pluripotent stem cells to cells of the hepatocyte lineage under special culture conditions.
Liver disease affects millions of people worldwide. Fulminant hepatic failure is the clinical term for an immediate and catastrophic cessation in liver function, usually leading to death within a matter of hours. Other forms of liver disease, such as chronic hepatitis and cirrhosis, involve an insidious and progressive failure of liver function, with grim effects on physiological well-being and long-term prognosis. In the United States, there are an estimated 300,000 hospitalizations each year for liver disease, and 30,000 deathsxe2x80x94with only about 4,500 donor livers available for transplant.
A healthy liver has a remarkable ability to regenerate itselfxe2x80x94but when this ability is compromised, the consequences are dire. An important challenge of modern medicine is to find a way to supplement the natural process of regeneration, and thereby restore liver function to affected patients.
Some early work has been done to identify liver progenitor cells in small animal models. Agelli et al. (Histochem. J. 29:205, 1997), Brill et al. (Dig. Dis. Sci. 44:364, 1999 and), and Reid et al. (U.S. Pat. No. 5,576,207) have proposed expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers. Michalopoulos et al. (Hepatology 29:90, 1999) report a system for culturing rat hepatocytes and nonparenchymal cells in biological matrices. Block et al. (J. Cell Biol. 132:1133, 1996) developed conditions for expansion, clonal growth, and specific differentiation in primary cultures of hepatocytes induced by a combination of growth factors in a chemically defined medium. It has been known for some time that mature rat liver cells derive from precursors (sometimes referred to as xe2x80x9chepatoblastsxe2x80x9d or xe2x80x9coval cellsxe2x80x9d) that have the capacity to differentiate into either mature hepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaro et al., Cancer Res. 58:514,1998; Germain et al., Cancer Res. 48:4909, 1988).
Unfortunately, a ready source of human hepatocytes for reconstitution therapy has not been identified. European Patent Application EP 953 633 A1 proposes a cell culturing method and medium for producing proliferated and differentiated human liver cells, apparently from donated human liver tissue. In most people""s hands, the replication capacity of human hepatocytes in culture has been disappointing. As a remedy, it has been proposed that hepatocytes be immortalized by transfecting with large T antigen of the SV40 virus (U.S. Pat. No. 5,869, 243).
A number of recent discoveries have raised expectations that stem cells may become a source of a variety of cell types and tissues for replacing those damaged in the course of disease, infection, or from congenital abnormalities. Various types of putative stem cells differentiate as they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, the liver, or the brain.
A particularly important development has been the isolation of two types of human pluripotent stem (hPS) cells from embryonic tissue. Pluripotent cells are believed to have the capacity to differentiate into most cell types in the body (R. A. Pedersen, Scientif. Am. 280(4):68, 1999). Early work on embryonic stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). However, monkey and human pluripotent cells have proven to be much more fragile, and do not respond to the same culture conditions as mouse embryonic cells. It is only recently that discoveries were made that allow primate embryonic cells to be obtained and cultured ex vivo.
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully culture embryonic stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and International Patent Application WO 98/43679). Both hES and hEG cells have the long-sought characteristics of human pluripotent stem (hPS) cells: they are capable of ongoing proliferation in vitro without differentiating, they retain a normal karyotype, and they retain the capacity to differentiate to produce all adult cell types.
Spontaneous differentiation of pluripotent stem cells in culture or in teratomas generates cell populations with a heterogeneous mixture of phenotypes, representing a spectrum of different cell lineages. In a number of applications, it is desirable for differentiated cells to be of a more homogeneous naturexe2x80x94both in terms of the phenotypes they express, and in terms of the types of progeny they can generate.
Accordingly, there is a need for technology to generate more homogeneous differentiated cell populations from pluripotent embryonic cells of primate origin, particularly those from humans.
This invention provides a system for efficient production of primate cells that have differentiated from pluripotent cells into cells of the hepatocyte lineage. Cultures of such cells have been obtained that are relatively enriched for characteristics typical of liver cells, compared with undifferentiated cells and cells that are committed to other tissue types.
One embodiment of the invention is a cell population obtained by differentiating primate pluripotent stem (pPS) cells in such a manner that a significant proportion of cells in the population have characteristics of cells of the hepatocyte lineage. Desirable characteristics are listed later in the description. The cells may demonstrate any or all of the following: antibody-detectable expression of xcex11-antitrypsin or albumin; absence of antibody-detectable expression of xcex1-fetoprotein; expression of asialoglycoprotein receptor at a level detectable by reverse PCR amplification; evidence of glycogen storage; evidence of cytochrome p450 or glucose-6-phosphatase activity; and morphological features of hepatocytes. Preferred cell populations have more of these hepatocyte characteristics in a greater proportion of the cells in the population. It is understood that the cells may replicate to form progeny, both during differentiation, and in subsequent manipulation. Such progeny also fall within the scope of the invention in all instances where not explicitly excluded.
Exemplary cells are obtained by differentiating embryonic stem (hES) cells obtained from cultures that originated from human blastocysts. The differentiated cells are generated by culturing the pPS cells in a growth environment that comprises a hepatocyte differentiation agent, such as n-butyric acid or other differentiation agent outlined in the disclosure. The differentiation agent can be added directly to undifferentiated pPS cells cultured with or without feeder cells. Alternatively, the pPS cells are allowed to differentiate into a mixed cell population (e.g., by forming embryoid bodies or by culture overgrowth), and the differentiation agent is added to the mixed population. What emerges is a less heterogeneous population, in which a substantial proportion of the cells have the desired phenotype. In some instances, the culture method also includes hepatocyte maturation factors such as those exemplified in the disclosure, which include solvents like DMSO, growth factors like FGF, EGF, and hepatocyte growth factor, and glucocorticoids like dexamethazone.
Another embodiment of the invention is a differentiated cell having characteristics of a cell of the hepatocyte lineage, which is either harvested from a differentiated cell population of this invention, or is the progeny of a cell harvested from such a population. Exemplary is a differentiated cell produced by providing a human pluripotent stem (hPS) cells in a growth environment essentially free of feeder cells; culturing the hPS cells in a medium containing a hepatocyte differentiation agent under conditions that produce a cell population enriched for cells with characteristic features of hepatocytes; and subsequently harvesting the differentiated cell from the enriched cell population.
Another embodiment of the invention is a method of treating human pluripotent stem (hPS) cells to obtain differentiated cells that can be maintained in an in vitro culture, by providing a culture of the hPS cells, and culturing the cells on a substrate in a culture medium containing a hepatocyte differentiation agent under conditions that permit enrichment of the differentiated cells. Beneficial techniques and reagents for use in the context of such methods are detailed later in the disclosure. Also embodied in the invention is a differentiated cell produced according to a method of this invention, particularly those having characteristics of cells of the hepatocyte lineage.
Yet another embodiment of the invention is a method of screening a compound for hepatocellular toxicity or modulation, comprising contacting a differentiated cell of this invention, and determining any phenotypic or metabolic changes in the cell that result. Another embodiment of the invention is a method of detoxifying a fluid such as blood, comprising contacting a differentiated cell of this invention with the fluid under conditions that permit the cell to remove or modify a toxin in the fluid. In this context, the differentiated cells described in this disclosure can be used as part of a liver support device, or for therapeutic administration for reconstituting hepatocellular function in an individual.
These and other embodiments of the invention will be apparent from the description that follows.